Genome-Wide Gene Expression Profiling of Nucleus Accumbens Neurons Projecting to Ventral Pallidum Using both Microarray and Transcriptome Sequencing

The cellular heterogeneity of brain poses a particularly thorny issue in genome-wide gene expression studies. Because laser capture microdissection (LCM) enables the precise extraction of a small area of tissue, we combined LCM with neuronal track tracing to collect nucleus accumbens shell neurons that project to ventral pallidum, which are of particular interest in the study of reward and addiction. Four independent biological samples of accumbens projection neurons were obtained. Approximately 500 pg of total RNA from each sample was then amplified linearly and subjected to Affymetrix microarray and Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD) transcriptome sequencing (RNA-seq). A total of 375 million 50-bp reads were obtained from RNA-seq. Approximately 57% of these reads were mapped to the rat reference genome (Baylor 3.4/rn4). Approximately 11,000 unique RefSeq genes and 100,000 unique exons were identified from each sample. Of the unmapped reads, the quality scores were 4.74 ± 0.42 lower than the mapped reads. When RNA-seq and microarray data from the same samples were compared, Pearson correlations were between 0.764 and 0.798. The variances in data obtained for the four samples by microarray and RNA-seq were similar for medium to high abundance genes, but less among low abundance genes detected by microarray. Analysis of 34 genes by real-time polymerase chain reaction showed higher correlation with RNA-seq (0.66) than with microarray (0.46). Further analysis showed 20–30 million 50-bp reads are sufficient to provide estimates of gene expression levels comparable to those produced by microarray. In summary, this study showed that picogram quantities of total RNA obtained by LCM of ∼700 individual neurons is sufficient to take advantage of the benefits provided by the transcriptome sequencing technology, such as low background noise, high dynamic range, and high precision

Protection genes in nucleus accumbens shell affect vulnerability to nicotine self-administration across isogenic strains of adolescent rat.

Classical genetic studies show the heritability of cigarette smoking is 0.4-0.6, and that multiple genes confer susceptibility and resistance to smoking. Despite recent advances in identifying genes associated with smoking behaviors, the major source of this heritability and its impact on susceptibility and resistance are largely unknown. Operant self-administration (SA) of intravenous nicotine is an established model for smoking behavior. We recently confirmed that genetic factors exert strong control over nicotine intake in isogenic rat strains. Because the processing of afferent dopaminergic signals by nucleus accumbens shell (AcbS) is critical for acquisition and maintenance of motivated behaviors reinforced by nicotine, we hypothesized that differential basal gene expression in AcbS accounts for much of the strain-to-strain variation in nicotine SA. We therefore sequenced the transcriptome of AcbS samples obtained by laser capture microdissection from 10 isogenic adolescent rat strains and compared all RNA transcript levels with behavior. Weighted gene co-expression network analysis, a systems biology method, found 12 modules (i.e., unique sets of genes that covary across all samples) that correlated (p<0.05) with amount of self-administered nicotine; 9 of 12 correlated negatively, implying a protective role. PCR confirmed selected genes from these modules. Chilibot, a literature mining tool, identified 15 genes within 1 module that were nominally associated with cigarette smoking, thereby providing strong support for the analytical approach. This is the first report demonstrating that nicotine intake by adolescent rodents is associated with the expression of specific genes in AcbS of the mesolimbic system, which controls motivated behaviors. These findings provide new insights into genetic mechanisms that predispose or protect against tobacco addiction

Nnat of the Pink module.

<p>The eigenvalue of the Pink module was significantly correlated with nicotine intake (A) but its correlation with food reward earned was not significant (B), suggesting behavioral specificity of the module. Nnat contributed strongly to the expression characteristics of the Pink module (C). The expression pattern of Nnat across the 10 strains measured by PCR showed significant correlation with nicotine intake (D). Strains: BN: Brown Norway, DA: Dark Agouti, F344: Fisher 344, Lew: Lewis, SHR: Spontaneous hypertensive rat, WKY: Wistar-Kyoto. For the F1 hybrids, the two letters representing the initials of the maternal and paternal strains were used.</p

WGCNA detected networks of correlated genes.

<p>(A) Weighted Gene Correlation Network Analysis (WGCNA) was applied to 12,639 genes. A total of 127 modules were identified in AcbS. Each module was named after a unique color assigned by the algorithm. Genes within each module were strongly correlated with each other across all the samples. (B) Modules significantly associated with any of the listed traits are plotted. Twelve modules were associated with stable levels of nicotine intake during last 3 d of SA (when nicotine intakes were stable). Of the 12 modules, nine were negatively correlated with nicotine intake, suggesting their expression in the AcbS protect against voluntary nicotine intake in adolescent rodents.</p